The present invention relates to peptides and proteins capable of desensitizing, in a specific manner, the great majority of subjects allergic to bee venom, and to compositions containing said peptides or proteins.
Immediate allergy to Hymenoptera (bee, wasp, hornet) affects 15 to 20% of the population, if prick tests are taken into account, but only 0.1 to 0.5% of the population is exposed to an anaphylactic-type accident (1, 2). This allergy is characterized by varying manifestations ranging from local swelling to systemic reactions such as urticaria, angioedema or anaphylactic shock. Given the suddenness of the stings, desensitization (specific immunotherapy or SIT) with venoms constitutes the preferred treatment for this allergy but is not without danger. Indeed, 13% of patients desensitized with bee venom and 5% in the case of wasp venom are victims of side effects (3). The search for a better tolerated and equally effective SIT is therefore necessary, in particular for bee venom.
The speed of the reactions observed in patients allergic to bee venom is characteristic of an immediate-type allergy which is mediated by IgEs specific for the constituents of the venom. The complex mechanism of these reactions is summarized below.
IgEs appear gradually under the repeated action of stings and before any symptom becomes apparent. Although the bee venom comprises numerous peptides and proteins, all the components do not appear to be allergenic (4). Melittin, for example, induces IgEs in only 30% of patients, whereas the proportion increases to more than 90% for phospholipase A2 (PLA2) which is, as a result, considered to be the major allergen (API m1). The protein sequence of bee venom phospholipase A2 (API m1) is illustrated in FIG. 1 (SEQ ID NO: 8); this sequence is deduced from that of the complementary cDNA (36).
IgEs possess the property of binding, via their Fc fragment, to receptors situated on the tissue mastocytes and the blood basophils. When the allergen forms a complex with the specific IgEs bound to the membrane of the basophils or mastocytes, it causes degranulation of the cells and the release of molecules which are responsible for the principal manifestations observed during an allergic accident. IgEs are not solely responsible for the allergy because although the IgE level is an indicator for the disease, it has no diagnostic value for the state of the patients. It is not rare for patients to have high IgE levels without showing symptoms. The appearance of IgEs in allergic patients results from the production of type TH2 cytokines such as IL-4, IL-5 and IL-13 and is inhibited by the synthesis of IFN-xcex3 (6).
It is mainly the CD4+T lymphocytes which produce these cytokines. Specific T cells which secrete more IL-4 than IFN-xcex3 are effectively found in allergic patients, whereas the T cells isolated from nonallergic subjects produce more IFN-xcex3 than IL-4.
The TH2-type CD4+ T lymphocytes specific for the venom components therefore actively participate in the appearance and the maintenance of the allergy.
To protect the subjects allergic to bee venom, it was proposed long ago to desensitize the allergic subjects by specific immunotherapy (or SIT). The immunological mechanisms of SIT which are responsible for the improvement in the patient""s condition remain poorly understood. The induction of specific IgGs and of an IgG4 subclass (7) as well as the generation of suppressive CD8+ T cells (8) were initially proposed to account for the efficacy of the treatment. However, it was more recently observed that during the desensitization to bee venom, the proliferation of the CD4+ T lymphocytes specific for the allergen decreases while the secretion of IL-4 and IL-5 decreases (9, 10) or is diverted toward the production of IFN-xcex3 (11). Very similar results were also obtained with pollen allergens (12). These observations appear to indicate that the improvement in the patient""s condition results from a peripheral tolerance or from a drift toward a TH1-type profile for the CD4+ T lymphocytes specific for the allergen. They are furthermore in agreement with experiments carried out in animals. It has indeed been shown for several antigens that under injection conditions similar to those used to desensitize (such as subcutaneous injection) the anergy of the T lymphocytes is induced while a high response is observed for the same antigens when they are injected in the presence of adjuvant (13, 14). All these experiments make it possible to consider the CD4+ T lymphocytes specific for bee venom as the target cells for immunotherapy.
The CD4+ T lymphocytes possess a rearranged T receptor which allows them to selectively recognize peptide fragments derived from the degradation of the antigen by the presenting cells and presented by the Major Histocompatibility Complex class II (MHC II) molecules (15). The determinants which these peptide fragments carry and which the T lymphocytes effectively recognize are called T epitopes.
During desensitization, it is these determinants which are recognized by the T lymphocytes and which therefore constitute the basic elements for the production of alternative molecules for specific immunotherapy.
It has indeed been observed in vivo in mice for the allergens Fel d1 (cat hair), Der p1 (acarian: Dermatophagoides pterissimus) and Bet v1 (birch pollen) that the nasal, oral or subcutaneous administration of peptides carrying T epitopes of these allergens inhibits the activation of the specific T lymphocytes (16-18) and modulates the allergic reaction (16, 18).
Ideally, the desensitizing molecules should possess all the epitopes of the allergen and be free of reactivity toward the IgEs, so as to avoid the risks of accidents.
Several types of molecule are already being studied, including in the context of bee venom allergy and consist either of peptide fragments or of modified proteins.
Peptide Fragments
Using an empirical approach, the use of peptide fragments (19) to desensitize allergic patients has been proposed on several occasions as an alternative to conventional specific desensitization, including for bee venom allergy (20, 21).
These fragments generally no longer possess reactivity toward IgEs, but they may also have lost their capacity to be recognized by T lymphocytes.
More recently, allergen peptide fragments were chosen on the basis of their capacity to stimulate T lymphocytes in allergic patients (22).
In the case of the major bee venom allergen (API m1), fragments 50-69 and 83-97 have been described as being active during a study comprising a single patient (23).
In a study comprising forty patients (24), it is fragments 45-62 and 81-92 and 113-124 which proved to be active. These three fragments are only T epitopes for 25 to 45% of the patients and the authors do not exclude the existence of other epitopes (24, 25). These three peptides are undergoing clinical trial and appear to give encouraging results (22). Muller et al. (22) have used them to desensitize five allergic patients whose T lymphocytes proliferate in the presence of these peptides. No serious systemic effect was observed and the patients became tolerant to bee stings. This demonstrates the benefit of using peptides for desensitizing, but does not make it possible to extend the use of these peptides to other patients.
Another trial using peptides was set up for the cat allergen (Fel d1) (26). Several applications relating to peptides from ragweed (WO 93/21321; WO 96/13589), from Japanese cedar pollen (WO 93/01213; WO 94/01560) and from ryegrass pollen (WO 94/21675; WO 94/16068) have been filed.
All the peptides described in these applications were chosen on the basis of the stimulation of T lymphocytes in a group of allergic patients.
The approach followed by these various authors (23, 24 and 30) is based on cellular tests and not on binding tests. The results observed show that the active peptides vary according to the patients. In the latter three studies, the peptides containing the zone 80-90 are those which are most often a T epitope. They also show that the lymphocytes of several patients are stimulated by peptides containing the C-terminal portion of API m1.
For example, Kxc3xa4mmerer et al. (30) propose, according to the same principle of the stimulation test, using long fragments of API m1, in particular fragment 90-134. However, these fragments are specific for certain patients and are not suitable for a significant set of patients because their selection does not take into account the HLA type of the patients.
The stimulation tests indeed make it possible to select peptides suitable for desensitizing a given patient (22), but do not make it possible to extend the use of these peptides to patients other than those for which they were produced.
Modified Proteins
Another alternative to the use of native allergens is that of allergens modified such that they no longer exhibit reactivity toward the IgEs specific for the allergen, while preserving their reactivity toward the T lymphocytes. As the IgEs are mainly directed against conformational epitopes, the loss of reactivity can be easily obtained by destroying the three-dimensional structure of the allergen, which does not modify by the T epitopes. That is what was done for mutants of Der f2 (27) and Der p2 (28) in which the disulfide bridges were broken. Another way of proceeding is to introduce into the allergen point mutations which affect the recognition of the IgEs without modifying the three-dimensional structure (29). The small number of mutations introduced is thought not to modify the T epitopes.
The large differences observed between studies indicate the interindividual variability of the T epitopes and obviously make the choice of desensitizing molecules difficult. Furthermore, these studies, which use only a group of allergic patients, do not make it possible to ensure that all the T epitopes are conserved in the desensitizing molecule.
Accordingly, the inventors set themselves the objective of providing a set of peptides capable of desensitizing the great majority of subjects allergic to bee venom. Such a set of peptides has the property of being effective in a large number of subjects, whereas the prior art peptides are active in one allergic subject but may be completely ineffective for another subject because the latter does not recognize the allergen by the same determinants.
To do this, the inventors have defined a relationship between the peptide sequences of the major bee venom allergen (API m1) and MHC class II molecules, both in the alleles of the HLA-DRB1 gene (1st gene), and the alleles of the DRB3, DRB4 and DRB5 genes (2nd gene), which are predominant in Caucasian populations. This relationship makes it possible, unexpectedly, to define molecules for desensitization and for preventive treatment of allergy to bee venom which takes into account the polymorphism of the MHC class II, in particular HLA-DR molecules, and which by virtue of their high specificity, induce better desensitization which results in a significantly reduced risk of accidents (shocks) during desensitization. This represents an additional advantage for the preventive use of such peptides.
The molecules of the major histocompatibility complex (MHC) class II (HLA II in man) are heterodimers expressed on presenting cells and present the T epitopes of the antigens to the CD4+ T lymphocytes. These molecules are capable of binding a large repertoire of peptides having very different sequences, which allows them to present several peptides per antigen to the T cells.
There are four different types of MHC II molecules per individual (2 HLA-DR, 1 HLA-DQ and 1 HLA-DP). The HLA-DR molecule, whose xcex2 chain is encoded by the DRB1 gene (1st gene) is the most highly expressed. There have currently been recorded more than 200 different alleles for DRB1, which define various antigens or types, as summarized in Table 1 below.
Each allele possesses its own binding properties. It therefore binds a repertoire of peptides specific to it and which differs from that for another allele, even on the same antigen. The broad specificity of the HLA II molecules and the existence of several isoforms and of a high polymorphism mean that many different fragments of the antigen can be presented to the T lymphocytes.
The frequencies of each allele (1st gene) are not identical and vary from one population to another (35):
the DRB1*1304 allele represents, on its own, 25.4% of the alleles in the Senegalese population against 0% in Germany and in Japan,
the DRB1*0301 allele is observed with a frequency of 10% in the Senegalese and the Germans but only at 0.4% in the Japanese,
in France, only seven alleles exceed 5%. They are the alleles DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 and DRB*1501, as illustrated in Table II below.
They represent, on their own, 63% of the French population. These same alleles are also the most abundant in the other Caucasian populations. Their frequencies vary from 53% (in Spain) to 82% (in Denmark). For the United States and Canada, they represent 58 and 55% of the population, respectively. They therefore represent, on their own, 53 to 82% of the alleles in Caucasian populations and are part of the various specificities of HLA-DR series.
The 2nd gene encodes the HLA-DRB3, -DRB4 and -DRB5 molecules which are HLA-DR molecules whose xcex2 chain is not encoded by the DRB1 gene. Although less well known than the molecules derived from the 1st gene, these HLA molecules are functional and are capable of presenting peptides to the T lymphocytes (56-59). Their main advantage for the immunotherapy is that alleles such as DRB3*0101 (9.2%), DRB4*0101 (28.4%) and DRB5*0101 (7.9%) are very frequent in the Caucasian population. They cover, on their own, 45% of the gene frequency. They are systematically associated with another HLA-DR molecule and can therefore complement its specificity. A strong binding disequilibrium exists between the 1st and 2nd gene, that is to say that a 2nd gene is very often associated with particular alleles of the 1st gene. The set of DR pseudogene and genes present on the same chromosome constitutes a DR haplotype (FIG. 5). Each haplotype is defined by the second DR molecule which characterizes it.
The role of the MHC II molecules in allergy was historically initiated by the discovery of the association between the level of IgE against a given allergen and certain alleles. These associations concern numerous allergens of low molecular mass such as Amb a5, Lol p3 and Amb a6 (31) which are allergens for which the relative risks are the greatest. These associations are not systematic or correspond to very low relative risks.
In the case of bee venom, it has been shown that the HLA-DR7 allele is more frequent in allergic patients than in the control population (32) whereas the HLA-DR4 allele is by contrast underrepresented in those allergic to bee venom (33).
The control of the IgE (and IgG) response by the class II molecules is even clearer in mice (34). The H-2d and H-2k mice are indeed good responders whereas the H-2b mice respond little or not at all to this allergen.
The subject of the present invention is peptides capable of desensitizing a subject allergic to bee venom, characterized in that they are selected from the group consisting of:
fragment (1) corresponding to positions P85-97 of the major bee venom allergen,
fragment (2) corresponding to positions P81-93 of the major bee venom allergen,
fragment (3) corresponding to positions P94-106 of the major bee venom allergen,
fragment (4) corresponding to positions P76-88 of the major bee venom allergen,
fragment (5) corresponding to positions P77-94 of the major bee venom allergen,
fragment (6) corresponding to positions P122-134 of the major bee venom allergen, and
the mutated fragments of said fragments (1) to (6) which exhibit an MHC class II molecule binding activity identical to or higher than those of said fragments (1) to (6).
Fragments (1) and (2) form group I; fragment (3) forms group II; fragments (4) and (5) form group III and fragment (6) forms group IV.
Such peptides comprise T epitopes or determinants which interact with one or more HLA-DR molecules derived from the DRB1 genes or other DRBs (DRB3, DRB4 and DRB5).
The present invention also includes the peptides as defined above, polymerized.
The site for binding of the peptides to the class II molecules is situated between the xcex1 helices of the xcex11 and xcex12 domains and forms a groove which is open at both ends. This opening allows the binding of peptides with varying sizes, in general from 13 to 25 amino acids. The anchoring of the peptides to the MHC II molecules occurs by means of hydrogen bonds between the backbone of the peptide and the amino acids of the groove and by means of residues accommodated by pockets for specificity. Five pockets, called P1, P4, P6, P7 and P9, correspond to the amino acid of the peptide which it accommodates, the first position being that which is in the first pocket, receive amino acids of the peptide and are composed of conserved or polymorphic residues. The polymorphic residues are responsible for various specificities between MHC II molecules. Since the binding site is open, two peptides binding one MHC II molecule may do so according to different modes, that is to say using different anchoring residues in their sequence.
Peptides P81-93 and P85-97, mutated in at least one residue, correspond to one of the pockets P1, P4, P6, P7 and P9 are in particular included in the invention.
In order to be able to introduce residues which preserve or increase the binding activity, the modes of interaction of the peptides P81-93 and P85-97 with respect to the HLA-DR molecules capable of binding them were studied. The approach chosen was to introduce into each position an alanine so as to evaluate the role of the side chain in the interaction or a lysine which is a basic and bulky amino acid. If necessary, a combination of mutations was introduced. By way of example, a reduction in activity caused by substitutions of phenylalanine 88 by a lysine or an alanine is observed in FIG. 6. Other reductions in activity are observed a positions situated at 3, 5 and 8 amino acids from phenylalanine 88. This activity profile corresponds to a mode of binding where positions F88, I91 T93 and Y96 are accommodated by the pockets P1, P4, P6 and P9, respectively, of the molecules HLA-DRB3*0101, HLA-DRB5*0101, HLA-DRB1*1301 and HLA-DRB1*0701. This mode of association was confirmed by molecular modeling for the complexes: P85-97/DRB3*0101, P85-97/DRB5*0101 and P81-93/DRB4*0101. All the results are given in FIG. 7. It is observed that on sequence 81-97, there are at least six modes of binding to the HLA-DR molecules. It is also observed that mode I is common to eight molecules whereas modes V and VI are specific to a single molecule.
The fact that it is possible to know exactly how the peptide is positioned on each HLA-DR molecule makes it possible to propose sequence modifications:
at positions P1, P4, P6, P7 and P9, it is possible to introduce modifications which are compatible with the known specificities of the HLA molecules (61). It is for example probable that tyrosine 87 can be changed by a phenylalanine without any loss of activity. On the other hand, the substitution of phenylalanine 88 by a tyrosine should not cause a loss of activity for, for example, the molecules DRB1*0101 and DRB1*0401 but should reduce the activity of the peptide for molecules such as DRB1*0301 or DRB3*0101.
it is also possible to increase the affinity of the peptide by modifying, in an optimum manner, residues P1, P4, P6, P7 and P9. This is what was done for residues 89 and 84. Residue 89 can be advantageously modified to leucine or to threonine (Table IX). In particular, the substitution by leucine increases the affinity of the peptide 85-97 by a factor of 10 and 12 for the molecules DRB3*0101 and DRB1*0301, respectively. In the case of DRB1*0301, this increase is explained by the fact that asparagine N89 at position P1 is not optimum for this molecule, unlike leucine (61). Likewise, the substitution of glycine 84 by an alanine increases the affinity by a factor of 5 for DRB1*1501. This increase is in agreement with the positioning of glycine 84 in P1 and ought to be even higher if a hydrophobic residue such as leucine or isoleucine is introduced. On the other hand, the substitution of aspartic acid D92 systematically reduces the affinity for DRB1*0301. This reduction in activity is in accordance with the anchoring role of aspartic acid in P4 for this molecule.
at positions P-2, P-1, P2, P3, P5, P8, P10 and P11, modifications may be introduced without fear of causing major losses in affinity.
The peptides N89L, N89T, D92N, D92L, D92T are analogs of the peptide P85-97A. The first letter corresponds to the initial amino acid, the number to its position in the Api m1 sequence and the second letter to the modification introduced. The data correspond to the ratios between the IC50 values for the analogs and that for the reference peptide P85-97A. Axe2x80x94sign means a loss of activity and the absence of a sign means an increase in activity.
The peptide P85-97A possesses an alanine at position 95 as a replacement for cysteine. This substitution does not cause any modification in activity regardless of the HLA-DR molecule.
In accordance with the invention, at least one of positions P1, P4, P6 and P9 of the fragments (1), with reference to FIG. 7 is mutated.
According to an advantageous feature of this embodiment, said fragments (1) comprise one of the following mutated amino acids: N89L, N89T, C95A, G84L, G84I.
Also in accordance with the invention, at least one of the positions P-2, P-1, P2, P3, P5, P8, P10 and P11 of the fragments (1), with reference to FIG. 7, is mutated.
The subject of the present invention is also desensitizing compositions for bee venom allergies, characterized in that they comprise:
at least one peptide selected from group A consisting of:
the peptides of group I, as defined above, and
the peptides consisting of fragments of at least 13 amino acids which are included in or comprise the fragment corresponding to positions P81-97 of the major bee venom allergen (API m1) and which bind at least to the HLA-DR molecules encoded by the HLA alleles DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301 and DRB1*1501 (molecules DR1, DR3, DR4, DR7, DR11, DR13 and DR2), with a binding activity  less than 1000 nM, and
at least one pharmaceutically acceptable vehicle.
Such compositions which are particularly suitable for the patient may be advantageously used preventively or curatively.
According to an advantageous embodiment of said compositions, they preferably consist of a mixture of peptides comprising:
at least one group A peptide as defined above and at least one other peptide selected from the following groups:
the peptides selected from group B consisting of:
the peptides of group II, as defined above, and
the peptides consisting of fragments of at least 13 amino acids which are included in or comprise the fragment corresponding to positions 94-106 of the major bee venom allergen (API m1) and which bind at least to the HLA-DR molecules expressed by the alleles DRB1*0101, DRB1*0401 and DRB1*1101 (molecules DR1, DR4 and DR11), with a binding activity  less than 1000 nM,
the peptides selected from group C consisting of:
the peptides of group III, as defined above, and
the peptides consisting of fragments of at least 13 amino acids which are included in or comprise the fragment corresponding to positions P76-94 of the major bee venom allergen and which bind at least to the HLA-DR molecules expressed by the alleles DRB1*0701, DRB1*1101 and DRB1*1501 (molecules DR7, DR11 and DR2), with a binding activity  less than 1000 nM, and
the peptides selected from group D consisting of:
the peptides of group IV, as defined above, and
the peptides consisting of fragments of at least 13 amino acids which are included in or comprise the fragment corresponding to positions P122-134 of the major bee venom allergen and which bind at least to the HLA-DR molecules expressed by the alleles DRB1*1101, DRB1*1301 and DRB1*1501 (molecules DR11, DR13 and DR2), with a binding activity  less than 1000 nM.
According to another embodiment of said compositions, they comprise, in addition, optionally the peptide corresponding to positions P18-30 and/or the peptide corresponding to positions P45-62 and/or the peptide corresponding to positions P57-74 and/or the peptide corresponding to positions P65-82 and/or the peptide corresponding to positions P111-123, of the major bee venom allergen, in accordance with Tables IIIa and IIIb below and/or the peptides including the abovementioned peptides.
These peptides are representative of the zones of interaction with the 2nd HLA-DR molecules. They are part of the most active peptides for a given zone and are as short as possible.
a: the cumulative frequencies between the alleles of various genes only make sense because the 2nd molecules behave, within the Caucasian population, like an identical allelic series.
Advantageously, in said compositions:
the group I peptides may be advantageously concatenated to form a single peptide (P81-97) and/or
the group III peptides may be advantageously concatenated to form a single peptide (P76-94) corresponding to positions P81-97 of the major bee venom allergen and/or
the group II and group III peptides may be advantageously concatenated to form a single peptide (P76-106) corresponding to positions P81-97 of the major bee venom allergen.
In general, the compositions according to the invention comprise at least one peptide including at least one of those described in Table III and which are suitable for the patient to be desensitized.
These desensitizing compositions are defined from the activities for binding to the HLA-DR molecules of the peptides which they comprise, from the frequency of alleles toward which they are active and from the complementarity of the zones for interaction or epitopes which said peptides carry.
For a patient for whom the HLA-DR molecules which they are carrying are not known, a composition according to the invention will be preferably used which comprises at least one group A peptide.
There may be added thereto, advantageously and in order to increase the number of epitopes: a group B peptide; a group C peptide; a peptide corresponding to the concatenation of a group B peptide and of a group C peptide and/or a group D peptide.
For the patients for whom the HLA molecules are known, there will be used either the peptide defined above, or a combination of peptides including at least those described in Tables IIIa and IIIb, which correspond to the alleles which the patient possesses.
The peptides included in said compositions were advantageously selected using an HLA-DR/peptide binding test comprising (i) incubating the purified HLA-DR molecules selected from those relating to more than 5% of a given population and in particular the HLA molecules DR1, DR3, DR4, DR7, DR11, DR13 and DR2, simultaneously with various concentrations of fragments of 13 to 18 amino acids which overlap and which completely cover the API m1 sequence and with a reagent R1 consisting of a peptide fragment combined with a nonradioactive marker, such as biotin and whose sequence is different from said peptides and is chosen such that it exhibits affinity toward the chosen HLA-DR molecule, such that it can be used at a concentration  less than 200 nM, (ii) transferring the complexes obtained on an ELISA-type plate, previously sensitized with an antibody specific for all the HLA-DR molecules, (iii) revealing the HLA-DR molecules/R1 reagent complexes, attached to the bottom of the plate by means of suitable conjugates, such as streptavidin-phosphatase and a fluorescent substrate, (iv) selecting the peptides comprising different epitopes, that is to say the most representative of the various zones of interaction between the major bee venom allergen and the HLA-DR molecules and (v) choosing the most suitable peptides as a function of the frequency of the alleles toward which they exhibit a binding activity  less than 1000 nM, corresponding to the concentration of this peptide which inhibits 50% of the binding of the reagent R1 (IC50).
These tests make it possible, unambiguously, to combine with each allele of the 1st gene or of the 2nd gene, the sequences of the fragments capable of binding thereto or on the contrary which do not bind thereto.
This approach makes it possible to define desensitizing compositions including peptides which bind to the largest number of different HLA-DR molecules and which can thus be advantageously desensitized for the majority of patients, even if their HLA molecules are not known.
This approach has, in addition, the advantage of allowing the selection of peptides which are significantly more specific with respect to most of the allergic subjects than the approaches seeking to select peptides on the basis of their capacity to stimulate T lymphocytes of allergic subjects.
Thus, the inventors have found that only some peptides have a binding activity with respect to several of the most frequent alleles in the Caucasian population in accordance with Tables IIIa and IIIb.
The subject of the present invention is also desensitizing compositions for bee venom allergies, characterized in that they comprise at least one modified bee venom phospholipase A2, in which the zones comprising the peptides as defined above are conserved and the zones outside the abovementioned zones are modified, such that they no longer exhibit reactivity toward the IgEs and at least one pharmaceutically acceptable vehicle.
Said zones are in particular modified by point mutation or deletion. Bee venom PLA2 is indeed capable of receiving numerous mutations and deletions (53, 54). It is therefore possible to obtain mutants no longer exhibiting reactivity toward IgEs, such as those obtained for Bet VI (55). For example, the bee PLA2 mutant in which the lysine at position 25 has been substituted is a lot less antigenic for apiarist sera than the native molecule (55).
The subject of the present invention is also the use of a modified bee venom phospholipase A2 in which the zones comprising the peptides as defined above are conserved and the zones outside the abovementioned zones are modified, such that they no longer exhibit reactivity toward the IgEs, for the preparation of a desensitizing composition for bee venom allergies.
The peptides which can be included in a desensitizing composition for bee venom allergies may be advantageously selected by a method which comprises:
(i) incubating the purified HLA-DR molecules selected from those relating either to less than 5% of a given population, that is to say those consisting of HLA-DRs other than the HLA molecules DR1, DR3, DR4, DR7, DR11, DR13 and DR2, or HLA-DR molecules from a given patient, simultaneously with various concentrations of fragments of 13 to 18 amino acids which overlap and which completely cover the API m1 sequence and with a reagent R1 consisting of a peptide fragment combined with a nonradioactive marker such as biotin and whose sequence is different from the peptides, as defined above (groups A to D) and is chosen so that it exhibits affinity toward the chosen HLA-DR molecule, such that it can be used at a concentration  less than 200 nM, (ii) transferring the complexes obtained on a microtiter plate, previously sensitized with an antibody specific for all the HLA-DR molcules, (iii) revealing the HLA-DR molecules/R1 reagent complexes, attached to the bottom of the plate by means of suitable conjugates, such as streptavidin-phosphatase and a fluorescent substrate, (iv) selecting the peptides comprising different epitopes, that is to say the most representative of the various zones of interaction between the major bee venom allergen and the HLA-DR molecules studied and (v) choosing the most suitable peptides as a function of the frequency of the alleles toward which they exhibit a binding activity  less than 1000 nM, corresponding to the concentration of this peptide which inhibits 50% of the binding of the reagent R1 (IC50).
The incubation conditions are specific to each HLA-DR molecule (incubation time, pH, reagent R1, HLA-DR or peptide concentration).
The reagent R1 is selected from the group consisting of the following sequences:
PKYVKQNTLKLAT (SEQ ID NO: 1), specific for the alleles DRB1*0101, DRB1*0401, DRB1*1101,
EAEQLRAYLDGTGVE (SEQ ID NO: 2), specific for the allele DRB1*1501,
AKTIAYDEEARGLE (SEQ ID NO: 3), specific for the allele DRB1*0301,
AAYAAAKAAALAA (SEQ ID NO: 4), specific for the allele DRB1*0701,
TERVRLVTRHIYNREE (SEQ ID NO: 5), specific for the allele DRB1*1301,
ESWGAVWRIDTPDKLTGPFT (SEQ ID NO: 6), specific for the alleles DRB1*1301, DRB3*0101, and
AGDLLAIETDKATI (SEQ ID NO: 7), specific for the alleles DRB1*0701 and DRB4*0101.
Other reagents R1 may be used, in particular those described in Southwood et al. (52).
To study the HLA-DR molecules (2nd gene), this requires appropriate pairs of biotinylated peptides which should bind the preparation at low concentration and be selective for one of the two molecules. More precisely, the binding of the biotinylated peptides should be effectively inhibited by their nonbiotinylated homolog, but not greatly disrupted by the nonbiotinylated form of the other peptide (Table X). It has been possible to find such peptides for each of the molecules DRB3*0101, DRB4*0101 or DRB5*0101.
Using these tests, the peptides of Api m1 which bind these molecules were defined. The same set of peptides as above was used: peptides of 18 amino acids which cover the sequence of the allergen and peptides of 13 residues which exhaustively cover particular zones. The results obtained are illustrated in Table X below.
The DRB5*0101 allele interacts with seven different regions of Api m1. Only four and five regions of Api m1 can bind the alleles DRB4*0101 and DRB3*0101, respectively. They are mainly situated in the central and C-terminal part of the allergen. Peptides P111-123 and 122-134 indeed binds the three second molecules. It is also advantageous to note that peptides common to the 1st and 2nd DR molecules, in particular the peptides P81-93 and P85-97, exist.
The selectivity of each peptide is evaluated by the IC50 of the nonbiotinylated peptide tested on each of the molecules. For each pair, the first peptide is that which is selective for the 1st DR molecule and the second for the 2nd DR molecule.
The sequences of the peptides are as follows: B1 21-36
(TERVRLVTRHIYNREE) (SEQ ID NO: 5) (62), LOL 191-210
(ESWGAVWRIDTPDKLTGPFT) (SEQ ID NO: 6) (63), YKL
(AAYAAAKAAALAA) (SEQ ID NO: 4) (64), HA 306-318
(PKYVKQNTLKLAT) (SEQ ID NO: 1) (65), A3 152-166
(EAEQLRAYLDGTGVE) (SEQ ID NO: 2) (65), E2/E168
(AGDLLAIETDKATI) (SEQ ID NO: 7).
Surprisingly, the method of selection according to the invention is suitable for any HLA-DR molecule.
The subject of the invention is also a kit for selecting peptides capable of desensitizing a subject allergic to bee venom, characterized in that it comprises various concentrations of fragments of 13 to 18 amino acids which overlap and which completely cover the API m1 sequence, a set of reagents R1 each consisting of a peptide fragment combined with a non-radioactive marker, such as biotin and whose sequence is different from the peptides as defined above (groups A to D) and is chosen such that it exhibits affinity toward the chosen HLA-DR molecule such that it can be used at a concentration  less than 200 nM and an antibody specific for all the HLA-DR molecules.